diversion out of the flow stream of large cells and even entire
organisms for collection. White papers describing applications
with Arabidopsis can be found at http://www.unionbio.com/
applications/notes.aspx?id¼20
1.7 Flow Cytometry
and Sorting of Plant
Homogenates
Flow cytometers were originally designed for the analysis of cells in
suspension. As previously mentioned, they are also suitable for
analysis of various other suspensions of non-cellular particles,
assuming these particles produce detectable light-scatter and/or
fluorescence signals. This means that one strategy for analyzing
complex plant tissues and organs is to convert them into homogenates of organelles, and use cytometry to classify these organelles
following staining with fluorochromes. This strategy has been particularly productive for characterization of the DNA contents of
nuclei, being first described in 1983 [5]. In contrast to the situation
with flow cytometry of mammalian cell suspensions, which benefits
from the observation that most of the objects that are detected
within the suspensions are cells, a different situation exists when
flow cytometry is applied to cellular homogenates, since the analysis
needs to detect a small, and sometimes very small, minority of the
general light-scattering objects, within a large background of
debris. In the measurement of nuclear DNA contents within arabidopsis homogenates, the nuclei comprise only 1–2% of the objects
actually detected in the biparametric fluorescence histograms
[24, 129, 130]. Importantly, it should be noted that this small
proportion may overstate the proportions of nuclei in the homogenate, since one also is required to pre-adjust discriminator settings
(a voltage offset) to eliminate the avalanche of light-scattering
signals resulting from subcellular debris smaller than the nuclei.
Difficulties occur when the optical properties are not known for the
objects of interest and the background debris. Further complications are associated with the presence in the homogenate of colored
autofluorescent organelles such as chloroplasts.
Despite these issues, in comparing the use of protoplasts or
homogenates as source samples for cytometric analyses of the cell
cycle and DNA contents, homogenates provide a number of advantages. For mammalian cells, cell cycle analyses and measurements of
genome sizes are routinely done using cell suspensions
[131]. Access of DNA-specific fluorochromes to the nuclei is
achieved either via prior fixation of the cells, or through use of
cell-permeant DNA fluorochromes such as Hoechst 33342. It also
helps that mammalian cells are generally translucent, produce only
minor amounts of autofluorescence, and are small in comparison to
the spot sizes of the focused lasers in the cytometer. In contrast,
plant cells can be highly pigmented and autofluorescent, which
adversely affects the consistency of illumination of individual protoplasts and of the quantitative measurement of the fluorescence
emitted by the nuclei within these protoplasts. The much larger
sizes of protoplasts compared to their nuclei also introduces
Flow Cytometry and Sorting in Arabidopsis
267
organisms for collection. White papers describing applications
with Arabidopsis can be found at http://www.unionbio.com/
applications/notes.aspx?id¼20
1.7 Flow Cytometry
and Sorting of Plant
Homogenates
Flow cytometers were originally designed for the analysis of cells in
suspension. As previously mentioned, they are also suitable for
analysis of various other suspensions of non-cellular particles,
assuming these particles produce detectable light-scatter and/or
fluorescence signals. This means that one strategy for analyzing
complex plant tissues and organs is to convert them into homogenates of organelles, and use cytometry to classify these organelles
following staining with fluorochromes. This strategy has been particularly productive for characterization of the DNA contents of
nuclei, being first described in 1983 [5]. In contrast to the situation
with flow cytometry of mammalian cell suspensions, which benefits
from the observation that most of the objects that are detected
within the suspensions are cells, a different situation exists when
flow cytometry is applied to cellular homogenates, since the analysis
needs to detect a small, and sometimes very small, minority of the
general light-scattering objects, within a large background of
debris. In the measurement of nuclear DNA contents within arabidopsis homogenates, the nuclei comprise only 1–2% of the objects
actually detected in the biparametric fluorescence histograms
[24, 129, 130]. Importantly, it should be noted that this small
proportion may overstate the proportions of nuclei in the homogenate, since one also is required to pre-adjust discriminator settings
(a voltage offset) to eliminate the avalanche of light-scattering
signals resulting from subcellular debris smaller than the nuclei.
Difficulties occur when the optical properties are not known for the
objects of interest and the background debris. Further complications are associated with the presence in the homogenate of colored
autofluorescent organelles such as chloroplasts.
Despite these issues, in comparing the use of protoplasts or
homogenates as source samples for cytometric analyses of the cell
cycle and DNA contents, homogenates provide a number of advantages. For mammalian cells, cell cycle analyses and measurements of
genome sizes are routinely done using cell suspensions
[131]. Access of DNA-specific fluorochromes to the nuclei is
achieved either via prior fixation of the cells, or through use of
cell-permeant DNA fluorochromes such as Hoechst 33342. It also
helps that mammalian cells are generally translucent, produce only
minor amounts of autofluorescence, and are small in comparison to
the spot sizes of the focused lasers in the cytometer. In contrast,
plant cells can be highly pigmented and autofluorescent, which
adversely affects the consistency of illumination of individual protoplasts and of the quantitative measurement of the fluorescence
emitted by the nuclei within these protoplasts. The much larger
sizes of protoplasts compared to their nuclei also introduces
Flow Cytometry and Sorting in Arabidopsis
267